PROJECT SUMMARY/ABSTRACT There is a global urgency to develop a protective vaccine against HIV-1. Although neutralizing antibodies (nAbs) can provide effective prophylaxis against HIV-1 acquisition, eliciting those that are broadly reactive against many antigenically diverse HIV-1 isolates has been the major challenge and it remains a critical roadblock to AIDS vaccine development. The primary objective of this proposal is to establish a vaccine strategy that can elicit broadly neutralizing antibodies (bnAbs) against the CD4 binding site (CD4BS) of HIV-1 gp120 with a long-term goal of developing a protective AIDS vaccine. We will evaluate a novel IPAS-RAM vaccine strategy (Incremental, Phased Antigenic Stimulation for Rapid Antibody Maturation), using a combination of rationally designed envelope constructs derived from eOD-GT6, MSC3, 426c gp120 core, and BG505 SOSIP gp140. This proposal is based on the scientific premise that vaccine formulation and immunization schedule are just as critical as the immunogens themselves in eliciting antibody responses. The major hypotheses to be tested are (1) antibody responses mounted against multiple antigenically-distinct immunogens presented to the immune system concurrently will be different from those against the same immunogens presented individually, one at a time; (2) in a multiple-immunogen environment, relative dosages of each immunogen can affect immunodominance of B-cell epitopes; and (3) the sequential order of which different antigens are presented to the immune system will affect antibody responses. The basic concept of the IPAS-RAM vaccine strategy is to prime the immune system using a germline-targeting small immunogen to stimulate a broad spectrum of Abs against the CD4BS, then selectively amplify those that bind the native structure by boosting with progressively more native immunogens. What makes our IPAS-RAM strategy superior to simple sequential immunizations is that the immune system is exposed to different, but related, immunogens simultaneously in a phased manner, such that B cells stimulated by a smaller immunogen can concurrently engage common epitopes on a larger, more native immunogen. Successful completion of this study would establish a novel vaccine strategy to induce bnAbs against HIV-1.